PROJECT SUMMARY The 200 Mammals Project is an NHGRI-funded project building a comparative genomics resource of unparalleled power for disease research, to greatly accelerate research focused on identifying causal variants for many human diseases. Since our launch in 2016, we have made and shared genome assemblies for over 130 new, extraordinarily diverse, mammalian species, and completed the largest ever multiple genome alignment. This alignment, with 240 species, is far bigger than even the ambitious goals of our original proposal. As a result, for the first time, we have the power to identify specific single nucleotides in the human genome as unusually conserved over the 100 million years of mammalian evolution. Here, we request one additional year of support for the 200 Mammals Project, scaled back from the annual commitment in the project's first three years. With salary support for the Broad Institute team alone, we will be able to complete development of, and disseminate, key resources and tools. This work is crucial to ensuring the funding committed to this project yields exceptional value for disease researchers. (1) Generate and validate sequence conservation scores across the human genome, as well as in the genomes of key model species. Deliverables: (1) Genome tracks in the UCSC browser annotating both constraint and acceleration for four genomes: human (hg38), chimpanzee (panTro6), mouse (mm10) and dog (canFam3); (2) A software tool that will enable researchers to generate such tracks for any of the 240 species in the alignment. (2) Provide resources and tools for using 200 Mammals conservation scores in human disease studies. Deliverables: (1) Measures of evolutionary conservation and acceleration at human SNPs as a new functional annotation; (2) Integration of SNP annotation into S-LDSC (genetic architecture of complex disease) and PolyFun (finemapping of complex disease loci); (3) Metrics defining improved value of the 200 Mammals conservation scores over prior functional annotations for different diseases and disease architectures. (3) Annotate protein-coding genes, transposable elements, and repeats in each of our new genome assemblies. Deliverables: For each assembly, we will provide (1) a catalog of gene locations determined using comparative analysis of our alignment; (2) a catalog of canonical repeats and transposable elements. (4) Identify the genomic basis of exceptional mammalian traits. Deliverables: A catalog of genomic loci associated with exceptional traits not seen in humans, including hibernation, tolerance of extreme temperatures, and tolerance of blood glucose variability.